Field of the Invention
The invention generally relates to nerve guides for use in nerve regeneration. In particular, the invention provides nerve guides which are formed from three dimensional (3D) arrays of highly aligned electrospun fibers that are oriented in parallel with the long axis of the seamless and cylindrically shaped constructs. Gaps and elongated spaces between the stacked fiber arrays provide channels for directed axonal growth.
Background of the Invention
A peripheral nerve is an enclosed, cable-like bundle of peripheral axons (long, slender projections of neurons, see FIG. 1). A nerve provides a common pathway for the electrochemical nerve impulses that are transmitted along each of the axons. Each nerve is a cordlike structure that contains many axons. These axons are often referred to as “(nerve) fibers”. Within a nerve, each axon is surrounded by a layer of connective tissue called the endoneurium. The axons are bundled together into groups called fascicles, and each fascicle is wrapped in a layer of connective tissue called the perineurium. Finally, the entire nerve is wrapped in a layer of connective tissue called the epineurium. Herein, “nerve” and “axon” may be used interchangeably.
After an injury, peripheral nerves can undergo an astounding degree of regeneration. When a nerve is severed, all signals distal to the injury site are immediately lost. Over time, downstream axons undergo Wallerian degeneration [1]. The surviving nerves of the proximal segment subsequently begin to undergo regeneration in response to soluble factors, many of which are produced by Schwann cells [2,3]. If the precipitating injury cleanly severs the nerve, treatment may be confined to a surgery that is designed to re-establish the continuity between the truncated stumps of the damaged nerve. In this surgery the proximal and the distal aspects of the perineural sheath are sutured together to form an end-to-end anastomosis. In more extreme injuries where a long segment of the nerve is crushed or completely lost, treatment is greatly complicated. Under these conditions a conduit, or nerve guide, is used to bridge the gap and direct the regenerating axons to grow towards the distal stump.
Nerve guides in the peripheral nervous system have a relatively long clinical history; these tubular constructs are designed to direct the natural processes that lead to regeneration [4,5]. A variety of natural and bioengineered materials have been used in this type of application, with mixed success [6]. Early synthetic guides consisted of a simple, hollow tube that provided little more than a protected environment [7]. With the hollow core design regenerating axons literally spill out of the proximal nerve stump and grow down the conduit. This not-so subtle “spilling effect” jumbles normal nerve topography (this term refers to the relative position of the individual axons to each other within the nerve) and greatly reduces the efficiency, and fidelity, of axon targeting.
Next generation peripheral nerve guides have been fabricated to contain signal molecules [8] and/or structural features [9] that are intended to provide guidance cues to the regenerating axons. Functional recovery with these constructs can be quite extensive, as long as the nerve guide is used to bridge a gap of less than about 10 mm in length. Once the injury gap exceeds this threshold, the regeneration process will be compromised to varying degrees. Typically, in these injuries only a limited number of axons will actually traverse the wound bed and the efficiency of targeting to the distal tissues is poor, resulting in limited functional recovery. Further exacerbating these complications, a series of irreversible degenerative changes begin to evolve in the distal tissues. As these degenerative changes become entrenched, the prospects of meaningful functional recovery are greatly diminished, even if a large number of axons are efficiently targeted to these sites.
Despite extensive and continued development, the autologous nerve segment represents the state of the art treatment for long defect nerve injuries. This tissue is highly anisotropic and its native architecture provides many potential channels to guide regenerating axons across the wound bed. However, use of this autologous tissue comes at the expense of donor site morbidity and results in the transplantation of a “nerve guide” that is packed with axonal fragments. These fragments must first degenerate before the regenerating axons can penetrate into the remaining endoneurium of the tissue. This degeneration process slows regeneration by initially impairing the penetration of the nascent axons into the autograft, a complication that clearly exacerbates the effects of long term muscle de-innervation and tissue atrophy.
In many ways there are similarities between peripheral nerve and the spinal cord. Both have a highly organized anisotropic structure and axons organized into specific topographical relationships. One clear difference that distinguishes spinal cord tissue from the peripheral nervous system is the observation that the spinal cord has axons that travel from the brain downward to the base of the spinal cord and out into the peripheral tissues as well as axons that travel from the peripheral tissues upwards towards the brain. For complete regeneration to occur after spinal cord injury both aspects of this “two way trafficking” of axons must be restored. Spinal cord injury (SCI), in both human insults and animal models, results in the liquefactive necrosis of the tissue in and around the lesion site. A fluid-filled cyst commonly emerges as the final consequence of this process (FIG. 2). Due to the lack of a solid substrate, this late-stage pathological endpoint represents a physical gap that impedes axonal regeneration and functional recovery. Additionally, astrocytes within the injury site proliferate, hypertrophy and begin to express chondroitin sulfate proteoglycans (CSPGs), which represent potent inhibitors to axon regeneration. At the neuronal level, axotomy of motor and sensory neurons results in the loss of trophic support by target tissue, which exasperates neuronal cell death. Additionally, the presence of myelin debris, as a result of oligodendrocyte death, acts as a potent inhibitor to axon regeneration in the spinal cord. Collectively, these cellular responses to spinal cord injury represent major impediments to axon regeneration and functional recovery.
Mono-therapies that address each of these obstacles to regeneration individually have resulted in only limited axonal regrowth and functional recovery. To date, no single intervention has been devised to collectively address all of the known obstacles to axon regeneration in the spinal cord.
Several studies have demonstrated that aligned arrays of electrospun fibers can provide the guidance cues necessary to induce axons and glial cells to express a highly polarized phenotype [10-13]. Despite these preliminary and encouraging results, it is difficult to fabricate a clinically relevant nerve guide using the conventional electrospinning process which uses a rapidly rotating target mandrel to produce aligned fibers. Conventional electrospinning systems are very effective at producing flat, 2D sheets with highly anisotropic fibers [14-16]. These constructs are easily amenable to experimentation in vitro, however, a 2D sheet is less acceptable, or adaptable, for use in vivo. A flat 2D sheet can be spun into a very thick structure and then cut into strips that resemble “square cylinders.” These structures are technically seamless, however, fiber alignment tends to degrade the thicker a sheet becomes when fibers are processed under conventional 2D electrospinning conditions. Cutting such a sheet also distorts the existing alignment along the cutting plane. Also, in conventional electrospinning systems fiber alignment as induced by a rotating target mandrel is far more dependent upon fiber diameter than in the air gap system. In part this is because air currents and complex electric fields induced by the rotation of the mandrel disturb the trajectory of the charged jet, fiber flight and fiber deposition onto the target, thereby limiting the extent of alignment that can be achieved. Air gap electrospinning does not require any mandrel movement to induce fiber alignment, making it possible to produce aligned fiber arrays even when the polymer concentration is at the minimal threshold necessary to produce fibers. This allows highly aligned constructs to be fabricated from individual fibers of less than 200 microns in average cross sectional diameter, a size scale far smaller than can be achieved in conventional systems. Despite these limitations, the efficacy of using the conventional electrospinning process to fabricate hollow, cylindrical nerve guides has been explored to some extent. In these experiments the electrospun fibers have been deposited onto a round, rotating mandrel. While the fibers of this type of construct can be induced to exhibit a considerable degree of alignment when produced under these conditions, the fibers, unfortunately, are deposited onto the target mandrel in a circumferential orientation (i.e. the axis of alignment is ˜90° off with respect to the resulting long axis of the hollow tube). While the nano-to-micron diameter fibers that form the wall of this type of construct do represent a barrier that reduces the risk of inflammatory cells penetrating into the hollow lumen of the guide (critical to the regeneration process [17,18]), this architectural pattern (i.e. the arrangement of electrospun fibers in a circumferential pattern) does not lend itself well to providing directional guidance cues to regenerating axons.
Other attempts at making nerve guides from electrospun (and other) types of fibers generally involve rolling sheets of material to form tubes. Unfortunately, the resulting tubes are hollow and thus fail to mimic the architecture of natural nerve growth. Further, a flat sheet that is rolled into a tube must, of necessity, have “seams” where the edges of the rolled sheets are surface exposed, e.g. on the wall of the hollow center of the tube and/or on the surface of the tube, resulting in discontinuities and possible weak connective points in the structure. In attempts to produce a more autologous graft-like structure aligned sheets of electrospun materials and films have been prepared and rolled tightly into more compact structures (32,33). (The final construct resembles a cinnamon breakfast roll where the electrospun sheets are represented by the dough, and the gaps or seams between the rolled sheets are represented by the sugar and cinnamon). While these structures are composed of “aligned fiber arrays” even these structures contain large seams (with respect to the size of the axons), no matter how tightly they may be rolled during fabrication. These seams represent a potential nexus for mechanical failure and the infiltration by unwanted interstitial fibroblasts and or inflammatory cells. Rolling a sheet can not truly integrate the fibers on the nano-scale that is necessary to make a uniform set of “pores”. The gaps represent large “circumferentially aligned longitudinal pores (seams)” for axons to grow along on the underlying aligned fibers. In a sense this type of design provides a larger “2D” surface area to guide the growth of axons that are growing along the seams of the rolled sheets.
U.S. Pat. No. 6,031,148 (Hayes) discloses nerve guides made from rolled sheets of material which have hollow centers.
U.S. Pat. No. 6,821,946 (Goldspink et al.) discloses administering growth factors to damaged nerves via a conduit of unidirectionally oriented fibers containing an alginate matrix. However, the conduits are rolled sheets which form a tube with a hollow center that contains seams.
U.S. Pat. No. 7,374,774 (Bowlin) teaches electrospun materials with various uses, e.g. as nerve guides. However, the nerve guides are formed by rolling sheets of fibers, and thus have a hollow center and once again contains seams as a consequence.
US patent application 2010/0047310 (Chen et al.) discloses nerve guides comprised of biodegradable, biocompatible electrospun material. However, the guides are made from sheets of several layers of conduits which are rolled into cylinders and are thus hollow.
U.S. Pat. No. 7,727,441 (Yost et al.) describes a tubular tissue scaffold which comprises a tube having a wall, wherein the wall includes biopolymer (collagen) fibrils that are aligned in a helical pattern around the longitudinal axis of the tube, and where the pitch of the helical pattern changes with the radial position in the tube wall. The scaffold is capable of directing the morphological pattern of attached and growing cells to form a helical pattern around the tube walls, but would not be suitable for use as a nerve guide, where the axons must grow straight down the guide and not wrap or change topology during growth.